Synaptic transmission depends on the transport of classical neurotransmitters from the cytoplasm, where they are synthesized and accumulate after reuptake from the extracellular space, into synaptic vesicles, which then undergo regulated exocytosis. To understand how transport into synaptic vesicles contributes to neurotransmitter release, the postsynaptic response and ultimately behavior, we have focused on the proteins responsible for this basic function of the nerve terminal. In previous work, we have identified two families of proteins responsible for the transport of cationic and neutral transmitters into synaptic vesicles. More recently, we have found that a protein previously suggested to mediate the Na+-dependent uptake of inorganic phosphate across the plasma membrane, in fact transports glutamate into synaptic vesicles. We will now use the identified vesicular transport proteins to pursue several goals. First, we will characterize further the ionic mechanisms of transport. Since the vesicular transport of neurotransmitters involves proton exchange and charge movement, we will use pH imaging and associated currents (in addition to radiotracer flux assays) to study the function of vesicle transporters that we have deliberately mislocalized to the plasma membrane, as well as related plasma membrane transporters. Second, we will assess the potential for regulation by studying transporter phosphorylation and membrane trafficking. Substantial work already indicates that phosphorylation regulates the membrane trafficking of certain transport proteins to specialized neurosecretory vesicles, including large dense core vesicles as well as synaptic vesicles. We will now identify the sorting events involved and use the motifs to characterize the sorting machinery. In particular, we will use the transporters to identify interacting proteins that may contribute to their regulation, their subcellular localization or their coordination with other events in the synaptic vesicle cycle. Third, we will manipulate the expression, localization and regulation of the transporters in vivo to assess the role of these mechanisms in synaptic transmission and behavior.